Polyubiquitin chains: polymeric protein signals

doi:10.1016/j.cbpa.2004.09.009

Current Opinion in Chemical Biology

Volume 8, Issue 6, December 2004, Pages 610-616

https://doi.org/10.1016/j.cbpa.2004.09.009 Get rights and content

The 76-residue protein ubiquitin exists within eukaryotic cells both as a monomer and in the form of isopeptide-linked polymers called polyubiquitin chains. In two well-described cases, structurally distinct polyubiquitin chains represent functionally distinct intracellular signals. Recently, additional polymeric structures have been detected in vivo and in vitro, and several large families of proteins with polyubiquitin chain-binding activity have been discovered. Although the molecular mechanisms governing specificity in chain synthesis and recognition are still incompletely understood, the scope of signaling by polyubiquitin chains is likely to be broader than originally envisioned.

Introduction

The conserved protein ubiquitin (Ub) regulates a host of intracellular processes through its enzymatic conjugation to other cellular proteins. The final step in the process of ubiquitination, which is catalyzed by a Ub-protein ligase (E3), results in the formation of an isopeptide bond between Ub's C-terminal glycine residue (G76) and a lysine residue of the target protein. Outcomes of this modification include destabilization of the target protein [1], altered protein trafficking [2] and functional modulation [3]. An important question in Ub biology concerns how this functional range is achieved. Polyubiquitin (polyUb) chains — polymers formed through Ub–Ub conjugation — occur within cells, can be linked to target proteins, and take on diverse structures due to the presence of seven lysine residues in Ub (Figure 1a). The existence of structurally distinct polyUb chains could represent one way to enhance diversity in Ub-dependent signaling. Here, we review the evidence germane to this hypothesis, focusing on recent findings and important unanswered questions. Ub also defines a growing family of Ub-like modifiers and stable Ub-like domains that function as signaling and interaction elements, respectively 4., 5.. However, none of the Ub-like proteins is known to signal as a polymer in vivo.

Section snippets

Historical background

PolyUb chains were discovered through mechanistic studies of Ub's function in proteasome proteolysis, a vital regulatory mechanism that has recently become a therapeutic target 1., 6.. A pioneering biochemical study showed that a single K48-linked polyUb chain is sufficient to target a model substrate to 26S proteasomes [7], and such K48-linked chains proved to be the principal proteasome delivery signal [8]. Since then, it has become clear that mono-Ub and polyUb chains generally signal

Assessing polyubiquitin chain structure

Because the functional outcome of polyubiquitination can be linkage (structure) specific, determination of the lysines involved in chain formation is important. It is also difficult — at present there are no diagnostic reagents (such as linkage-specific antibodies) for use in linkage determination. The reigning methods for linkage assignment are mutagenesis and mass spectrometry. Mutagenic approaches include introducing specific lysine mutations and constructing a single-lysine version of Ub

Structural and conformational attributes of polyubiquitin chains

Function-relevant features of Ub's compact 3D structure include a hydrophobic surface patch formed by L8, V70 and I44, and a flexible C-terminus ending with G76 (Figure 1a). A combination of the hydrophobic effect and the electrostatic potential caused by the positive charges (on K6, R42, K48, H68 and R72, surrounding the hydrophobic patch) are likely to be important for Ub's interaction with multiple factors. In fact, many of these residues show perturbations upon mono/poly-Ub binding to

An expanding repertoire of polyubiquitin signals

K63-linked chains are now known to signal in four pathways: DNA damage tolerance [28], the inflammatory response [3], protein trafficking [2], and ribosomal protein synthesis [29]. In the first case, chain signaling is focused on the DNA polymerase processivity factor PCNA (proliferating cell nuclear antigen). PCNA trimerizes to encircle the template DNA during replication, serving as a platform for the binding of accessory factors whose access is coordinated by several mechanisms, including

Functional hypotheses

Is there a structural basis for the functional diversity of polyUb chains? An obvious model following from the structural features of Ub discussed above is that the linkage-dependent conformation of a polyUb chain determines its function either by presenting a linkage-specific pattern of hydrophobic sites or by restricting the chain's ability to adopt certain conformations. The Ub–Ub isopeptide linker could also contribute, with the aliphatic side chain of lysine (stripped of its charge)

(Poly)ubiqutin-binding domains: an expanding repertoire of signal transducers?

The past several years have seen the discovery of a remarkable collection of small Ub-interacting domains (Table 1), many of which probably serve as downstream receptors of monoUb and polyUb signals 2., 4.. This model clearly applies in the Ub-dependent sorting of membrane proteins, where the recognition of mono-Ub modifications by UIM (Ub interacting motif), CUE (coupling of ubiquitination to ER degradation), and UBA (Ub-associated) domains regulates trafficking decisions [2].

PolyUb chains

Conclusions

It is attractive to make a conceptual analogy between different polyUb chains and different phosphate modifications: just as there are kinases that phosphorylate serine versus tyrosine, and structural domains that selectively recognize the two phospho-amino acids, so are there E3 enzymes that generate differently-linked polyUb chains, and structural motifs that recognize distinct chains in a selective manner. Structure-specific polyUb depolymerizing enzymes — the functional analogs of

Update

Two recent papers describe a deubiquitinating enzyme that acts on K63-linked chains attached to specific target proteins. This enzyme blunts IKK activity and thus, downregulates inflammatory signaling 51., 52.. Interestingly, the linkage specificity is not intrinsic: when acting on free polyubiquitin chains, the enzyme prefers the K48 linkage 51., 52.. Another recent paper describes small molecules that bind K48-linked chains in preference to K29- and K63-linked chains [53]. These ‘ubistatins’

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

We are grateful to J Bender, R Cohen and M Hochstrasser for critical comments on the manuscript. Work in our laboratories is funded by grants from the NIH.

References (53)

C.M. Pickart
Back to the future with ubiquitin
Cell
(2004)
L. Sun et al.
The novel functions of ubiquitination in signaling
Curr Opin Cell Biol
(2004)
A. Buchberger
From UBA to UBX: new words in the ubiquitin vocabulary
Trends Cell Biol
(2002)
J. Adams
The development of proteasome inhibitors as anticancer drugs
Cancer Cell
(2004)
H. Nishikawa et al.
Mass spectrophotometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase
J Biol Chem
(2004)
R. Varadan et al.
Solution conformation of Lys⁶³-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling
J Biol Chem
(2004)
R.S. Kang et al.
Solution structure of a CUE-monoubiquitin complex reveals a conserved mode of ubiquitin binding
Cell
(2003)
W.I. Sundquist et al.
Ubiquitin recognition by the human TSG101 protein
Mol Cell
(2004)
K.E. Sloper-Mould et al.
Distinct functional surface regions on ubiquitin
J Biol Chem
(2001)
W.J. Cook et al.
Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2)
J Biol Chem
(1992)

H.D. Ulrich

Degradation or maintenance: actions of the ubiquitin system on eukaryotic chromatin

Eukaryot Cell

(2002)

J. Spence et al.

Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain

Cell

(2000)

P. Stelter et al.

Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation

Nature

(2003)

A.P. VanDemark et al.

Structural basis of ubiquitylation

Curr Opin Struct Biol

(2002)

F. Wu-Baer et al.

The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin

J Biol Chem

(2003)

K. Lindsten et al.

Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion protein degradation substrate that blocks proteasomal degradation

J Cell Biol

(2002)

Y. Saeki et al.

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Biochem Biophys Res Commun

(2004)

R.M. Hofmann et al.

27936-27943: In vitro assembly and recognition of Lys-63 polyubiquitin chains

J Biol Chem

(2001)

S. Raasi et al.

Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A

J Mol Biol

(2004)

T.D. Mueller et al.

Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions

J Mol Biol

(2002)

Wilkinson CRM et al.

Proteins containing the UBA domain are able to bind multi-ubiquitin chains

Nat Cell Biol

(2001)

P.C. Evans et al.

Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity

Biochem J

(2004)

A. Kovalenko et al.

The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination

Nature

(2003)

Miller SLH et al.

Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation

J Biol Chem

(2004)

Boone DL, Turer EE, Lee EG, Ahmad R-C, Wheller MT, Tsui C, Hurley P, Chien M, Chai S, Hitotsumatsu O et al.: The...

L. Hicke et al.

Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins

Annu Rev Cell Dev Biol

(2003)

Cited by (864)

Snakehead vesiculovirus hijacks SH3RF1 for replication via mediating K63-linked ubiquitination at K264 of the phosphoprotein
2024, International Journal of Biological Macromolecules
Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.
Betulinic acid, a major therapeutic triterpene of Celastrus orbiculatus Thunb., acts as a chemosensitizer of gemcitabine by promoting Chk1 degradation
2023, Journal of Ethnopharmacology
Celastrus orbiculatus Thunb., also called as oriental bittersweet vine or climbing spindle berry, a traditional Chinese herbal medicine has been used to treat a spectrum of painful and inflammatory diseases for centuries. Explored for their unique medicinal properties, C.orbiculatus offers additional therapeutic effects on cancerous diseases. The effect of single-agent gemcitabine on survival has not long been encouraging, combination therapies provide patients multiple chances of benefit for improved clinical response.
This study aims at expounding the chemopotentiating effects and underlying mechanisms of betulinic acid, a primary therapeutic triterpene of C. orbiculatus in combination with gemcitabine chemotherapy.
The preparation of betulinic acid was optimized using ultrasonic-assisted extraction method. Gemcitabine-resistant cell model was established by induction of the cytidine deaminase. MTT, colony formation, EdU incorporation and Annexin V/PI staining assays were used to evaluate cytotoxicity, cell proliferation and apoptosis in BxPC-3 pancreatic cancer cell line and H1299 non-small cell lung carcinoma cell line. Comet assay, metaphase chromosome spread and γH2AX immunostaining were applied for DNA damage assessment. Western blot and co-immunoprecipitation was used to detect the phosphorylation and ubiquitination of Chk1. Mode of action of gemcitabine in combination with betulinic acid was further captured in BxPC-3-derived mouse xenograft model.
We noticed that the extraction method had an impact on the thermal stability of C. orbiculatus. Ultrasound-assisted extraction at room temperature in shorter processing time could maximize the overall yields and biological activities of C. orbiculatus. The major constituent was identified as betulinic acid, and the pentacyclic triterpene represented the prominent anticancer activity of C. orbiculatus. Forced expression of cytidine deaminase conferred acquired resistance to gemcitabine, while betulinic acid displayed equivalent cytotoxicity toward gemcitabine-resistant and sensitive cells. A combination therapy of gemcitabine with betulinic acid produced synergistic pharmacologic interaction on cell viability, apoptosis and DNA double-strand breaks. Moreover, betulinic acid abrogated gemcitabine-triggered Chk1 activation by destabilizing Chk1 loading via proteasomal degradation. The combination of gemcitabine and betulinic acid significantly retarded BxPC-3 tumor growth in vivo compared to single-agent gemcitabine treatment alone, accompanied with reduced Chk1 expression.
These data provide evidence that betulinic acid is a potential candidate for chemosensitization as a naturally occurring Chk1 inhibitor and warrants further preclinical evaluation.
Deubiquitinase CYLD regulates excitatory synaptic transmission and short-term plasticity in the hippocampus
2023, Brain Research
The fate of proteins is determined by the addition of various forms of polyubiquitin during ubiquitin-mediated proteasomal degradation. Cylindromatosis (CYLD), a K63-specific deubiquitinase, is enriched in postsynaptic density fractions of the rodent central nervous system (CNS), but the synaptic role of CYLD in the CNS is poorly understand. Here we show that CYLD deficiency (Cyld^−/−) results in reduced intrinsic hippocampal neuronal firing, a decrease in the frequency of spontaneous excitatory postsynaptic currents and a decrease in the amplitude of field excitatory postsynaptic potentials. Moreover, Cyld^−/− hippocampus shows downregulated levels of presynaptic vesicular glutamate transporter 1 (vGlut1) and upregulated levels of postsynaptic GluA1, a subunit of the AMPA receptor, together with an altered paired-pulse ratio (PPR). We also found increased activation of astrocytes and microglia in the hippocampus of Cyld^−/− mice. The present study suggests a critical role for CYLD in mediating hippocampal neuronal and synaptic activity.
Biosynthesis of long polyubiquitin chains in high yield and purity
2023, Analytical Biochemistry
As one of the most prevalent protein post-translational modifications, ubiquitin modification plays a momentous role in regulating varied cellular functions. Different polyubiquitin linkage types have diverse effects on cell signaling. However, compared with short ubiquitin chains, the preparation of long ubiquitin chains remains difficult and expensive to purchase commercially. In this study, we constructed an enzyme library of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase E3, which are specific for synthesizing K63, K48, and M1 linked polyubiquitin chains. We demonstrate that these distinctly linked polyubiquitin chains could be synthesized and purified with high yield and purity. More importantly, this method can synthesize longer ubiquitin chains, the longest can reach more than fifteen ubiquitin molecules, which provides great convenience for ubiquitin-related structural and functional studies.
Progress in targeting PTEN/PI3K/Akt axis in glioblastoma therapy: Revisiting molecular interactions
2023, Biomedicine and Pharmacotherapy
Glioblastoma (GBM) is one of the most malignant cancers of central nervous system and due to its sensitive location, surgical resection has high risk and therefore, chemotherapy and radiotherapy are utilized for its treatment. However, chemoresistance and radio-resistance are other problems in GBM treatment. Hence, new therapies based on genes are recommended for treatment of GBM. PTEN is a tumor-suppressor operator in cancer that inhibits PI3K/Akt/mTOR axis in diminishing growth, metastasis and drug resistance. In the current review, the function of PTEN/PI3K/Akt axis in GBM progression is evaluated. Mutation or depletion of PTEN leads to increase in GBM progression. Low expression level of PTEN mediates poor prognosis in GBM and by increasing proliferation and invasion, promotes malignancy of tumor cells. Moreover, loss of PTEN signaling can result in therapy resistance in GBM. Activation of PTEN signaling impairs GBM metabolism via glycolysis inhibition. In contrast to PTEN, PI3K/Akt signaling has oncogenic function and during tumor progression, expression level of PI3K/Akt enhances. PI3K/Akt signaling shows positive association with oncogenic pathways and its expression similar to PTEN signaling, is regulated by non-coding RNAs. PTEN upregulation and PI3K/Akt signaling inhibition by anti-cancer agents can be beneficial in interfering GBM progression. This review emphasizes on the signaling networks related to PTEN/PI3K/Akt and provides new insights for targeting this axis in effective GBM treatment.
A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination
2024, Nature Communications

View all citing articles on Scopus

View full text

Polyubiquitin chains: polymeric protein signals

Introduction

Section snippets

Historical background

Assessing polyubiquitin chain structure

Structural and conformational attributes of polyubiquitin chains

An expanding repertoire of polyubiquitin signals

Functional hypotheses

(Poly)ubiqutin-binding domains: an expanding repertoire of signal transducers?

Conclusions

Update

References and recommended reading

Acknowledgements

Cell

Curr Opin Cell Biol

Trends Cell Biol

Cancer Cell

J Biol Chem

J Biol Chem

Cell

Mol Cell

J Biol Chem

J Biol Chem

Eukaryot Cell

Cell

Nature

Curr Opin Struct Biol

J Biol Chem

J Cell Biol

Biochem Biophys Res Commun

J Biol Chem

J Mol Biol

J Mol Biol

Nat Cell Biol

Biochem J

Nature

J Biol Chem

Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins

Annu Rev Cell Dev Biol